WO2021171088A1 - Nanovecteurs pour l'administration de molécules à des types de cellules cliniquement pertinents - Google Patents

Nanovecteurs pour l'administration de molécules à des types de cellules cliniquement pertinents Download PDF

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Publication number
WO2021171088A1
WO2021171088A1 PCT/IB2021/000104 IB2021000104W WO2021171088A1 WO 2021171088 A1 WO2021171088 A1 WO 2021171088A1 IB 2021000104 W IB2021000104 W IB 2021000104W WO 2021171088 A1 WO2021171088 A1 WO 2021171088A1
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nanocapsule
alkaline earth
earth metal
cell
metal carbonate
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PCT/IB2021/000104
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English (en)
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Albert MUSLIMOV
Alexander GONCHARENKO
Yana TARAKANCHIKOVA
Kirill LEPIK
Igor SERGEEV
Mikhail Trofimov
Oleksii PELTEK
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<<Qr.Bio>>, Limited Liability Company
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Publication of WO2021171088A1 publication Critical patent/WO2021171088A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes

Definitions

  • the present invention relates to the preparation of nanoparticles and/or nanocapsules, compositions comprising such nanoparticles and/or nanocapsules, and their use in delivery of molecules (e.g., nucleic acids, peptides, proteins, or small molecules) to various cell types, particularly clinically relevant cell types.
  • molecules e.g., nucleic acids, peptides, proteins, or small molecules
  • DNA- and messenger RNA (mRNA)-based therapies are currently changing the biomedical field, the delivery of genetic material remains the key problem preventing the wide introduction of these methods into the clinical practice.
  • mRNA messenger RNA
  • mRNA is an unstable biomolecule that mediates the translation of genetic information from genes encoded in DNA to proteins located throughout the cell.
  • the physical and biological characteristics of mRNA allow for its use as a safe genetic material for gene-based therapy approaches (Yin, H. et al., Nat. Rev. Genet. 2014, 15 (8), 541-555; Quabius, E. S. et al., N Biotechnol. 2015, 32 (1), 229-235).
  • the application of mRNA as a template does not require nuclear localization for gene expression and allows rapid protein expression even in nondividing and hard-to-transfect cells (e.g. T-cells and hematopoietic stem cells) with no risk of genomic integration.
  • mRNA an attractive molecule for cancer vaccination and immunotherapy (Lundstrom, K. Futur. Sci. OA 2018, 4 (5), FSO300) as well as for genome editing approaches (Deering, R. P. Expert Opin. Drug Deliv. 2014, 11 (6), 885-899).
  • Another advantage of mRNA as genetic material is its predictable and consistent protein expression kinetics, especially compared to DNA transfection (Lee, J. et al., Synthetic Messenger RNA and Cell Metabolism Modulation pp 111-125; Yamamoto, A. et al., Eur. J. Pharm. Biopharm. 2009, 71 (3), 484-489; Leonhardt, C. et al., Nanomedicine Nanotechnology, Biol. Med.
  • RNA vaccination in cancer and infectious diseases RNA vaccination in cancer and infectious diseases
  • RNA vaccination in cancer and infectious diseases RNA vaccination in cancer and infectious diseases
  • cytokines for cancer immunotherapy and autoimmunity control
  • Naked plasmid DNA is another type of nucleic acid that holds promise for use in therapeutic approaches because of its ease of handling and safety.
  • nucleic acids require delivery systems in order to be transported through the cell membrane and to be protected against enzymatic degradation.
  • Viral vectors have been used as mRNA and DNA carriers but have disadvantages, such as potential immunologic side effects and toxicity, vector-size limitations and extremely high cost for production in the clinical setting (Yin, H. et al., Nat. Rev. Genet. 2014, 15 (8), 541-555; Thomas, C. E. et al., Nat. Rev. Genet. 2003, 4 (5), 346-358).
  • Non-viral strategies such as electroporation, gene gun and sonoporation have been more thoroughly investigated as nucleic acids delivery systems (Tavernier, G. et al., J. Control.
  • liposomes compared to other common carriers, for example, lipid-based nanoparticles
  • delivery efficiency and biocompatibility are their high penetrating ability, delivery efficiency and biocompatibility.
  • they since they have low loading efficiency, especially for RNA molecules (Kooijmans, S. A. A. et al., J. Control. Release 2013, 172 (1), 229-238), and also because of the complicated manipulations of the formation of liposomes and nanovesicles (Lunavat, T. R. et al., Biomaterials 2016, 102, 231-238) are costly and time consuming procedures, a system that shares the benefits, but lacking the disadvantages of liposomes, could be an attractive alternative.
  • the present disclosure provides, among other things, nanoparticles and/or nanocapsules that can be used for delivery of molecules (e.g., nucleic acids, peptides, proteins, or small molecules) to various cell types.
  • molecules e.g., nucleic acids, peptides, proteins, or small molecules
  • a method of preparing an alkaline earth metal carbonate nanoparticle comprising mixing a first solution comprising a carbonate source, a stabilizer, and a solvent with a second solution comprising an alkaline earth metal salt.
  • the alkaline earth metal salt is a water soluble salt. In some embodiments, the alkaline earth metal salt is a chloride salt.
  • the carbonate source comprises a water soluble carbonate ion.
  • the carbonate source is a water soluble carbonate or hydrocarbonate.
  • the carbonate source is sodium hydrocarbonate (NaHCCh).
  • the stabilizer is an organic or inorganic phosphate, an organic or inorganic sulfonate, or an organic or inorganic sulphate. In some embodiments, the stabilizer is an organic phosphate. In some embodiments, the stabilizer is a phosphoric acid ester. In some embodiments, the stabilizer is adenosine triphosphate (ATP).
  • ATP adenosine triphosphate
  • the solvent in the first solution comprises water and optionally one or more water miscible solvents.
  • the solvent in the second solution comprises water and optionally one or more water miscible solvents.
  • the solvent in both the first and the second solutions comprises water and optionally ethanol.
  • the solvent comprises water and ethanol at a ratio of about 100: 0 to about 50: 50. In some embodiments, the solvent comprises water and ethanol at a ratio of about 50: 50.
  • the final concentration of the alkaline earth metal salt after mixing is between about 0.1M to about 3M. In some embodiments, the final concentration of the alkaline earth metal salt after mixing is between about 1.8M to about 2M. In one embodiment, the final concentration of the alkaline earth metal salt after mixing is about 1.875M.
  • the final concentration of the carbonate source after mixing is between about 6 mM to 40 mM. In some embodiments, the final concentration of the carbonate source after mixing is between about 6 mM to 10 mM. In one embodiment, the final concentration of the carbonate source is about 7.5 mM.
  • the final concentration of the stabilizer after mixing is between about 1 mM to 40 mM. In some embodiments, the final concentration of the stabilizer after mixing is between about 1 mM to 5 mM. In some embodiments, the final concentration of the stabilizer is about 1.25 mM.
  • the concentration ratio of the stabilizer and the carbonate source is from about 1:1 to about 1:6. In some embodiments, the concentration ratio of the stabilizer and the carbonate source is about 1:6. [0019] In some embodiments, the method further comprise adding gelatin to the solution after mixing. In some embodiments, gelatin has been heated to about 90-100°C. In some embodiments, gelatin is added to a concentration of about 90-100 g/L in the final solution. In one embodiment, gelatin is added to a concentration of about 96 g/L in the final solution.
  • the alkaline earth metal carbonate is selected from CaCCh, MgCCh, and BaCCh. In some embodiments, the alkaline earth metal carbonate is CaCCh. In some embodiments, the CaCCh nanoparticle is selected from a vaterite particle, a calcite particle, an aragonite particle, and a particle of amorphous CaCCh. In some embodiments, the CaCCh nanoparticle is a vaterite nanoparticle. In some embodiments, the alkaline earth metal carbonate is MgCCh.
  • the method further comprises precipitating the alkaline earth metal carbonate nanoparticle.
  • precipitating the alkaline earth metal carbonate nanoparticle is achieved by centrifugation.
  • the method further comprises stabilizing the alkaline earth metal carbonate nanoparticle by resuspending the alkaline earth metal carbonate nanoparticle in a phosphate solution.
  • the phosphate solution comprises a phosphoric acid salt, and/or a derivative thereof.
  • the phosphate solution comprises sodium triphosphate.
  • the pH of the phosphate solution is not less than 6.
  • the concentration of sodium triphosphate is between about 1 mg/ml to about 20 mg/ml. In some embodiments, the concentration of sodium triphosphate is between about 5 mg/ml to about 10 mg/ml. In some embodiments, the concentration of sodium triphosphate is about 7 mg/ml.
  • the method further comprises washing the alkaline earth metal carbonate nanoparticle before and/or after the stabilizing step. In some embodiments, the method further comprises resuspending the alkaline earth metal carbonate nanoparticle in a phosphate solution.
  • the phosphate solution comprises sodium triphosphate. In some embodiments, the concentration of sodium triphosphate is between about 0.01 mg/ml to about 0.5 mg/ml. In some embodiments, the concentration of sodium triphosphate is about 0.28 mg/ml.
  • the first solution further comprises a cargo and the cargo co precipitates with the alkaline earth metal carbonate nanoparticle.
  • the method further comprises adsorbing a cargo onto the alkaline earth metal carbonate nanoparticle.
  • the cargo is adsorbed onto the alkaline earth metal carbonate nanoparticle by contacting the alkaline earth metal carbonate nanoparticle with a solution comprising the cargo.
  • the cargo is adsorbed onto the alkaline earth metal carbonate nanoparticle by freezing-induced loading method.
  • the present disclosure provides an alkaline earth metal carbonate nanoparticle produced by the preparation method described herein.
  • the present disclosure provides a preparation of alkaline earth metal carbonate nanoparticles produced by the preparation method described herein, wherein at least 90% of the nanoparticles have a diameter of about 50 nm to about 300 nm. In some embodiments, at least 90% of the nanoparticles have a diameter of about 50 nm to about 150 nm.
  • the present disclosure provides a method of preparing a nanocapsule having a diameter of about 50 nm to about 500 nm comprising a) coating an alkaline earth metal carbonate nanoparticle having a diameter of about 50 nm to about 150 nm with one or more shell layers, each shell layer comprising at least one biodegradable material comprising two or more electronegative and/or electropositive groups, and optionally adsorbing a cargo to the one or more shell layers; and b) optionally, removing the alkaline earth metal carbonate nanoparticle.
  • the nanocapsule has a diameter of about 50 nm to about 300 nm. In some embodiments, the nanocapsule has a diameter of about 100 nm to about 200 nm.
  • the alkaline earth metal carbonate nanoparticle used in step (a) is produced by the preparation method described herein.
  • step (a) the one or more shell layers are added without a washing step between the addition of layers.
  • step (b) removing the alkaline earth metal carbonate nanoparticle is achieved by contacting the alkaline earth metal carbonate nanoparticle with an acid or sodium EDTA solution.
  • the two or more electronegative groups are selected from sulfonic acid, sulfuric acid, carboxylic acid, phosphoric acid, polyphosphoric acids, and phenolic hydroxyl, and combinations thereof.
  • the biodegradable material comprising two or more electronegative groups is selected from dextran sulfate, poly(4-vinylphenol), poly(sodium 4- styrenesulfonate), fucoidan, polyethylene glycol, bovine serum albumin, and tannic acid.
  • the two or more electropositive groups are selected from primary amines or their onium cations, secondary amines or their onium cations, tertiary amines or their onium cations, quaternary ammonium compounds, and cyclic amines or their onium cations, and combinations thereof.
  • the biodegradable material comprising two or more electropositive groups is selected from polyacrylamide, poly-l-lysine, poly-l-arginine, poly(allylamine hydrochloride), poly-l-ornithine, and chitosan.
  • the cargo comprises one or more of a nucleic acid, a peptide, a protein, or a small molecule.
  • the present disclosure provides a nanocapsule produced by the preparation method described herein.
  • the present disclosure provides a nanocapsule having a diameter of 50 nm to 500 nm comprising: a) a core comprising an alkaline earth metal carbonate nanoparticle; b) one or more shell layers coating the core comprising at least one biodegradable material comprising two or more electronegative and/or electropositive groups; and c) a cargo.
  • the alkaline earth metal carbonate is selected from CaCCb, MgCCb, and BaCCb. In some embodiments, the alkaline earth metal carbonate is CaCCb. In some embodiments, the CaCCb particle is selected from a vaterite particle, a calcite particle, an aragonite particle, and a particle of amorphous CaCCb. In some embodiments, the CaCCb particle is a vaterite particle. In some embodiments, the alkaline earth metal carbonate is MgCCb.
  • the present disclosure provides a hollow nanocapsule having a diameter of 50 nm to 500 nm comprising: a) one or more shell layers comprising at least one biodegradable material comprising two or more electronegative and/or electropositive groups, and b) a cargo.
  • the nanocapsule has a diameter of 50 nm to 300 nm. In some embodiments, the nanocapsule has a diameter of 100 nm to 200 nm.
  • the two or more electronegative groups are selected from sulfonic acid, sulfuric acid, carboxylic acid, phosphoric acid, polyphosphoric acid, and phenolic hydroxyl, and combinations thereof.
  • the biodegradable material comprising two or more electronegative groups is selected from dextran sulfate, poly(4-vinylphenol), poly(sodium 4- styrenesulfonate), polyethylene glycol, bovine serum albumin, and tannic acid.
  • the two or more electropositive groups are selected from primary amines or their onium cations, secondary amines or their onium cations, tertiary amines or their onium cations, quaternary ammonium compounds, and cyclic amines or their onium cations, and combinations thereof.
  • the biodegradable material comprising two or more electropositive groups is selected from polyacrylamide, poly-l-lysine, poly-l-arginine, poly(allylamine hydrochloride), poly-l-ornithine, and chitosan.
  • the at least one biodegradable material is selected from an oligopeptide, a polypeptide, an oligosaccharide, a polysaccharide, a glycopeptide, a glycolipid, a natural nucleic acid, an artificial nucleic acid, a polyphenol, a synthetic biodegradable polymer, and a biodegradable cross-linking agent, wherein the biodegradable material comprises two or more ionizable functional groups.
  • the at least one biodegradable material is an oligopeptide or a polypeptide comprising two or more ionizable amino acids selected from arginine, ornithine, lysine, histidine, aspartic acid, glutamic acid, and combinations thereof.
  • the at least one biodegradable material is an oligosaccharide or a polysaccharide comprising a monomer selected from glucose, fructose, ribose, galactose, and glucuronic acids and combinations thereof.
  • the oligosaccharide or polysaccharide comprises a sulfo-, a phospho-, an amino-, a carboxy-, or an amido-derivative of the monomer and combinations thereof.
  • the at least one biodegradable material is a synthetic biodegradable polymer selected from a polyester, a polyamine, polydiacetylene (PDA), polyethylene glycol (PEG), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), polyvinyl alcohol (PVA), polyhydroxybutyrate (PHB), polyvinylpyrrolidone (PVP), and derivatives and co-polymers thereof.
  • the biodegradable cross-linking agent is glutaraldehyde.
  • the cargo comprises one or more of a nucleic acid, a peptide, a protein, and/or a small molecule.
  • the cargo comprises a nucleic acid selected from a single-stranded DNA, a double-stranded DNA, a mini circle DNA, an oligodeoxynucleotide (ODN), an RNA, and synthetic analogs and derivatives thereof, or a protein-nucleic acid complex, and combinations thereof.
  • the RNA is selected from interfering RNA (RNAi) molecules, dsRNA, RNA polymerase III transcribed RNAs, messenger RNA (mRNA), antisense nucleic acids, and oligonucleotides.
  • RNAi molecule is an siRNA molecule or an shRNA molecule.
  • the protein-nucleic acid complex is a protein-nucleic acid complex associated with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/nuclease gene system.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the protein-nucleic acid complex associated with CRISPR/nuclease gene system is gRNA-Cas9 or gRNA-Cpfl.
  • the cargo comprises a peptide or a protein.
  • the cargo comprises a genome editing protein, a functional or immunogenic peptide, an antibody or antibody fragment, an enzyme, a receptor or receptor fragment, or a secreted factor.
  • the cargo comprises a nucleic acid encoding a genome editing protein, a functional or immunogenic peptide, an antibody or antibody fragment, a receptor or receptor fragment, an enzyme, or a secreted factor.
  • the genome editing protein is selected from a CRISPR protein, a zinc finger nuclease (ZFN), and a transcription activator-like effector nuclease (TALEN).
  • the antibody or antibody fragment is selected from a human antibody, a chimeric antibody, a bispecific antibody, a trifunctional antibody, a single chain variable fragment (scFv), a Fab, a Fab', a F(ab')2, and a Fv fragment.
  • the receptor or receptor fragment is selected from a T cell receptor, a TCR variable domain, and a chimeric antigen receptor (CAR).
  • the enzyme is selected from a transposase, an RNAse, a DNAse, a peptidase, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, a protease, a lipase, a nuclease, a carbohydrase, a phosphatase, a sulphatase, a neuraminidase, an esterase, a kinase, a glycosyl transferase, an oxidase, a reductase and a transaminase.
  • the secreted factor is a cytokine, or a growth factor.
  • the cargo comprises a small molecule.
  • the small molecule is selected from an antineoplastic drug, an immunosuppressant, a tyrosine kinase inhibitor, a demethylating agent, a histone deacetylase (HDAC) inhibitor, an indoleamine 2,3- dioxygenase (IDO) inhibitor, a three prime repair exonuclease 1 (TREX1) inhibitor, a deoxyribonuclease II (DNAse II) inhibitor, an immune modulator, a proteasome inhibitor, an antimicrobial agent, enzyme inhibitor, and intracellular immune response (AIM2, cGAS, TLR, STING) inhibitor, an oligodeoxynucleotide.
  • HDAC histone deacetylase
  • IDO indoleamine 2,3- dioxygenase
  • TREX1 three prime repair exonuclease 1
  • the small molecule is an antineoplastic drug selected from doxorubicin, vinblastin, etoposide, cisplatin, monomethyl auristatin E, and ozogamycin.
  • the small molecule is an immunosuppressant selected from tacrolimus, sirolimus, ciclosporin, and fmgolimod.
  • the small molecule is a tyrosine kinase inhibitor selected from dasatinib, ibrutinib, ruxolitinib, tofacitinib, axitinib, sorafenib, idelalisib, venetoclax, and erlotinib.
  • the small molecule is a demethylating agent which is decitabine. In some embodiments, the small molecule is a HDAC inhibitor which is vorinostat. In some embodiments, the small molecule is an IDO inhibitor which is epacadostat. In some embodiments, the small molecule is an immune modulator selected from cridanimod, dimeric amidobenzimidazole, lenalidomide, and pomalidomide. In some embodiments, the small molecule is a proteasome inhibitor selected from ixosamib and carfilzomib.
  • the small molecule is an antimicrobial agent selected from meropenem, tigecycline, amphotericin b, ganciclovir, foscamet, and tenofovir.
  • the small molecule is an intracellular immune response inhibitor selected from N- (4-Ethylphenyl)-N’-lH-indol-3-yl-urea (H-151) and N-(4-iodophenyl)-5-nitro-2- furancarboxamide (C-176).
  • the cargo is embedded in the alkaline earth metal carbonate nanoparticle core.
  • the cargo is adsorbed onto the alkaline earth metal carbonate particle core and coated by the one or more shell layers.
  • the cargo is encapsulated by the one or more shell layers.
  • the cargo is adsorbed onto the one or more shell layers.
  • the cargo is embedded in the one or more shell layers.
  • the present disclosure provides a composition comprising a plurality of the nanocapsules described herein and a pharmaceutically acceptable carrier, wherein at least 90% of the nanocapsules have a diameter of 50 nm to 500 nm. In some embodiments, at least 90% of the nanocapsules have a diameter of 100 nm to 200 nm.
  • the present disclosure provides a method of delivering a cargo to a target cell of a subject comprising administering to the subject an effective amount of the nanocapsules described herein or the composition described herein.
  • the target cell is a mammalian cell.
  • the target cell is a primary stem cell, a primary mesenchymal stem cell, a tumor cell, a cell forming tumor microenvironment, a hematopoietic stem cell, an induced pluripotent stem cell, an embryonal stem cell, a fetal stem cell, an egg cell, a spermatozoan cell, a germ layer cell, a T-cell, a B-cell, an NK-cell, a monocyte, a macrophage, a fibroblast, a neuron, an epithelial cell, a retinal stem cell, a light-sensing cell, a leukocyte or a progenitor thereof, an erythrocyte or a progenitor thereof, or a hepatocyte.
  • the epithelial cell is selected from an intestinal epithelium cell, a retinal pigmented epithelial (RPE) cell, an esophageal epithelial cell, a glandular epithelial cell, a bladder epithelium cell, a prostate epithelial cell, a mesothelium cell, an epidermis cell, an endothelium cell, an alveolar epithelium cell, a germinal epithelium cell, a mucosal epithelium cell, a conjunctival epithelium cell, a glomerular epithelium, a respiratory epithelium cell, and an epithelial stem cell.
  • RPE retinal pigmented epithelial
  • the nanocapsule is administered to the subject intratumorally, orally, sublingually, buccally, rectally, vaginally, via a gastric tube, subcutaneously, epicutaneously, transdermally, intravenously, intramuscularly, intraocularly, intraperitoneally, intratesticulary, intracerebrally, nasally, by inhalation, epidurally, or via extra-amniotic administration.
  • the subject is an experimental or veterinary animal.
  • the subject is human.
  • Figures 1A-1B depict an exemplary process for the preparation of an exemplary nanocapsule of the present invention, and its use in delivering a cargo to a target cell.
  • Figure 1A shows phase I of the process which includes the synthesis of the template nanoparticle, preparation of the-mRNA (GFP) loaded poly-L-arginine hydrochloride (PARG) complex and mRNA (GFP) loaded nanocapsule.
  • Figure IB shows phase II of the process which includes target cell transfection using the synthetic mRNA (GFP) loaded nanocarriers.
  • Figures 2A-2C show qualitative and quantitative comparison of different synthesis types for evaluating changes CaCCh nanoparticle sizes.
  • Figure 2A shows SEM images for morphology particles characterization.
  • Figure 2B shows DLS distribution for each CaCCh spheroid particles synthesis.
  • Figures 2C show graphical comparison and numerical estimate of particle size depending on synthesis conditions.
  • Figures 3A-3D show characterizations of CaCCh nanoparticles.
  • Figure 3A illustrates XRD pattern of CaCCh polymorphs (Va-Vaterite, Ca-Calcite, Ar- Aragonite). Vertical lines show data from the AMCSD datasets.
  • Figure 3B shows results from Raman spectroscopy.
  • Figure 3C is a TEM image of the CaCCh nanoparticles.
  • Figure 3D shows particle size distribution as analyzed by DLS.
  • Figure 4 is a graph showing changes in zeta potential with different number of layers during layer-by-layer coating.
  • Zero layer comprises bovine serum albumin (BSA) fluorescein isothiocyanate (FITC)
  • first layer comprises poly-L-arginine hydrochloride (PARG)
  • second layer comprises dextran sulfate (DEXS)
  • third layer comprises poly-L-arginine hydrochloride (PARG).
  • Figure 5A-5D illustrate SEM images and DLS distribution of CaCCh particles before TPP treatment ( Figures 5A, 5D) and after TPP treatment ( Figures 5B, 5D) for particles dissagregation.
  • Figures 6A-6B show carbonate particle morphology (SEM) and size distribution (DLS) after polymer layers covering.
  • Figures 7A-7B show magnetic separation results for T-cells. Magnetic separation was used to obtain a pure population of human lymphocytes from peripheral blood apheresis product.
  • Figures 8A-8D show flow cytometry and confocal analysis of T-cells treated with different amounts of FITC-labeled nanocapsules.
  • Figures 9A-9B show evaluation of mRNA and plasmid DNA delivery of GFP into the cells (T-lymphocytes) by confocal laser scanning microscopy (CLSM) and flow cytometry after 24 hours.
  • CLSM confocal laser scanning microscopy
  • Figure 10 is a schematic showing additional features of an exemplary nanocapsule of the present invention. DETAILED DESCRIPTION
  • the present disclosure is based on an unexpected discovery that low nanosized nanoparticles and nanocapsules can be generated using improved synthesis methods, and the nanoparticles and/or nanocapsules can be used as highly efficient and safe carriers to deliver molecules (e.g., nucleic acids, peptides, proteins, or small molecules) to various cell types, including clinically relevant cell types and/or hard-to-transfect cell types.
  • molecules e.g., nucleic acids, peptides, proteins, or small molecules
  • nanocarriers are actively internalized by up to 99% of primary T-lymphocytes and exert minimal toxicity with the viability of >90%. These nanocarriers mediate more efficient transfection when compared with the standard electroporation method (90% vs.
  • these polymeric nanocarriers can be used in serum contained basic culture medium without special conditions and equipment, thus can be introduced in clinical development.
  • the term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range.
  • the allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.
  • alkaline earth metal refers to a chemical element in Group 2 (Ila) of the periodic table.
  • Alkaline earth metals include beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), and radium (Ra).
  • an “alkaline earth metal carbonate” refers to the carbonate (CCb) form of an alkaline earth metal.
  • the alkaline earth metal carbonate is calcium carbonate (CaCCb), magnesium carbonate (MgCCb), or barium carbonate (BaCCb).
  • nanoparticle and “nanocapsule” are art-recognized and refer to structures that are less than about one micron (1000 nm) in diameter.
  • a nanoparticle having a core-shell structure is referred to herein as a “nanocapsule”.
  • template are denoted herein as particles of alkaline earth metal carbonates which can be removed by chemical ways.
  • the term “cargo” encompass any type of biological active substances integrated into nanoparticles or nanocapsules.
  • nucleic acid encompass both DNA and RNA unless specified otherwise.
  • nucleotide encompass both DNA and RNA unless specified otherwise.
  • protein or “peptide” as used herein encompasses all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP- ribosylation, pegylation, biotinylation, etc.).
  • modified proteins e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP- ribosylation, pegylation, biotinylation, etc.
  • gene refers to a DNA sequence that encodes through its template or messenger RNA a sequence of amino acids characteristic of a specific peptide, polypeptide, or protein.
  • gene also refers to a DNA sequence that encodes an RNA product.
  • gene as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5' and 3' ends.
  • transfection means the introduction of a “foreign” (i.e., extrinsic or extracellular) nucleic acid into a cell using recombinant DNA technology.
  • genetic modification means the introduction of a “foreign” (i.e., extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, for example, but not limitation, a protein or enzyme coded by the introduced gene or sequence.
  • the introduced gene or sequence may also be called a “cloned” gene, “transgene”, or “foreign” gene or sequence, may include regulatory or control sequences operably linked to the polynucleotide, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery.
  • the gene or sequence may include nonfunctional sequences or sequences with no known function.
  • a host cell that receives and expresses introduced DNA or RNA has been “genetically modified” or “genetically engineered”.
  • the DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or from a different genus or species.
  • biodegradable means that the materials degrades or breaks down into its component subunits, or digestion, e.g., by a biochemical process, of the material into smaller (e.g., non-polymeric) subunits.
  • the term “diameter” is art-recognized and is used herein to refer to either of the physical diameter or the hydrodynamic diameter.
  • the diameter of an essentially spherical particle may refer to the physical or hydrodynamic diameter.
  • the diameter of a nonspherical particle may refer preferentially to the hydrodynamic diameter.
  • the diameter of a non-spherical particle may refer to the largest linear distance between two points on the surface of the particle.
  • the diameter of the particles typically refers to the average diameter of the particles.
  • Particle diameter can be measured using a variety of techniques in the art including, but not limited to, dynamic light scattering. Unless indicated otherwise, the terms “size” and “diameter”, when referring to a particle, such as a nanoparticle or nanocapsule, are used interchangeably.
  • a composition containing nanoparticles or nanocapsules may include particles of a range of particle sizes.
  • the particle size distribution may be uniform, e.g., within less than about a 20% standard deviation of the mean volume diameter, and in other embodiments, still more uniform, e.g., within about 10%, 8%, 5%, 3%, or 2% of the median volume diameter.
  • the term “particle” as used herein refers to any particle formed of, having attached thereon or thereto, or incorporating a therapeutic, diagnostic or prophylactic agent, optionally including one or more polymers, liposomes, micelles, or other structural material.
  • a particle may be spherical or nonspherical.
  • a particle may be used, for example, for delivering a molecule to a target cell.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient, or vehicle with which the composition is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
  • the pharmaceutical carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin.
  • an “effective amount” refers to an amount effective to treat a disease, disorder, and/or condition, or to bring about a recited effect.
  • an amount effective can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art.
  • the term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host.
  • an “effective amount” generally means an amount that provides the desired effect.
  • patient refers to mammals, including, without limitation, human and veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, primates, etc.) and experimental animal models.
  • subject is a human.
  • ranges recited herein also encompass any and all possible subranges and combinations of subranges thereof, as well as the individual values making up the range, particularly integer values.
  • a recited range e.g., concentration ranges or particle sizes
  • Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths.
  • each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
  • the technology illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein.
  • Nanoparticles are particles in the nanometer size range, whereas microparticles are particles in the micrometer size range. Both types of particles may be used as a carrier for drug delivery. However, the difference in size between micro- and nanoparticles has numerous effects. It is important that some of the distinctions between particles of different sizes may not hold across classes of particles. Nanometer particles can load a higher amount of delivered material due to higher surface area and porous structure, unlike micron particles.
  • nanoparticles In nanoparticles, a greater proportion of drugs can be released by diffusion. Likewise, water can penetrate into the smaller particles more rapidly, which can result in an increased rate of release and generally in more rapid release kinetics. In addition, in polymers that are degraded hydrolytically, water penetration can result in particle deterioration, which can further accelerate drug release. Smaller particles are more likely to aggregate. Also, surfaces of nanoparticles can be functionalized to optimize binding to specific receptors. A smaller particle can have a better binding for a unit of particle mass than a larger one. Control of loading and release of genetic material allows calculation of the optimal concentrations of the substance to achieve the desired effect.
  • Particles can be internalized by the cells via endocytosis processes, which includes phagocytosis and pinocytosis.
  • Phagocytosis is a means of uptaking materials about 2-10 pm in diameter (Tabata Y, Ikada Y.1988. Macrophage phagocytosis of biodegradable microspheres composed of L-lactic acid/glycolic acid homo- and copolymers. J Biomed Mater Res 22:837-858, which is herein incorporated by reference in its entirety), and can be apply for relatively few cell types, such as macrophages, neutrophils, and dendritic cells.
  • Particle size difference is also very important for the system in vivo.
  • Microparticles may concentrate at a local tissue after injection. For example, 60 pm particles injected to the sciatic nerve were still found in quantity at the site of injection more than 8 weeks later (Kohane DS, Lipp M, Kinney RC, Anthony DC, Louis DN, Lotan N, Langer R.2002 Biocompatibility of lipid- protein-sugar particles containing bupivacaine in the epineurium. J Biomed Mater Res 59(3):450- 459, which is incorporated herein by reference in its entirety). The difference between micro- and nanoparticles is well seen in the abdominal cavity. Microparticles injected into the peritoneum of mice remained there for at least two week.
  • nanoparticles are generally too small to cause embolic phenomena, and can circulate throughout the vasculature, while large microparticles can embolize vessels with the same diameter
  • the circulation time of such particles can be greatly increased by surface modification, such as rendering the surface hydrophilic by PEGylation (Harris JM, Martin NE, Modi M.200E Pegylation: a novel process for modifying pharmacokinetics. Clin Pharmacokinet 40(7):539-551, which is incorporated herein by reference in its entirety).
  • any organic and inorganic nanoparticles that possess one or more of the following properties can be applied.
  • the preferred properties of template suitable for use in the present disclosure include: a) a diameter of less than about 50 nm; b) possiblity for cargo (e.g., nucleotide) adsorption onto the surface or intercalation into the particle core; c) possibility for particle material to interact with positively or negatively charged polyelectrolytes; d) high adsorption capacity; and/or e) possibility of particle material biodegradation (e.g., by physico-chemical actions).
  • cargo e.g., nucleotide
  • the present disclosure provides a method of preparing an alkaline earth metal carbonate nanoparticle comprising mixing a first solution comprising a carbonate source, a stabilizer, and a solvent with a second solution comprising an alkaline earth metal salt.
  • the alkaline earth metal carbonate is CaCCb.
  • CaCCb precipitates from aqueous solution enabling three anhydrous polymorphs (calcite, aragonite, and vaterite), two hydrated forms (hexahydrate ikaite and monohydrate), and an amorphous phase.
  • polymorphs calcium carbonate particles are actively used in a variety of applications due to higher solubility in water and unique mechanical, physical, and chemical properties, low toxicity, and biological inertness.
  • Precipitated calcium carbonate particles can act as abrasives, absorbents, anticaking agents, buffers, fillers, colorants, and emulsion stabilizers improving rheology, physical robustness, and visual appearance of products (PCT Application Pub. No. WO2013165600, which is incorporated herein by reference in its entirety).
  • Spherical vaterite particles are classified as spherulites or framboids with regard to the interior (Zhou, G.-T. et ah, Eur. J. Mineral. 2010, 22 (2), 259-269, which is incorporated herein by reference in its entirety) have a slightly charged surface and porous structure that predetermine their major advantage over other morphological forms of vaterite for encapsulation purposes (Yashchenok, A. et ah, J. Mater. Chem. B 2013, 1 (9), 1223, which is incorporated herein by reference in its entirety).
  • the control over the size and morphology of MeCCb particles is of particular importance to fully realize the benefits of these crystals in drug delivery.
  • the particles of MeCCb have many properties that make them suitable for preparation of drug delivery carriers.
  • the MeCCb nanoparticle is a CaCCb vaterite particle, a calcite particle, an aragonite particle, or a particle of amorphous CaCCb.
  • the CaCCb nanoparticle is a vaterite nanoparticle. All crystal phases of calcium carbonate as calcite, aragonite and amorphous calcium carbonate can be applied as a template.
  • methods of the present invention involve addition of gelatin and glycerol which decrease the calcium carbonate solubility (Trushina, D. B.; Bukreeva, T. V.; Antipina, M. N.
  • gelatin has been heated to about 90-100°C. In one embodiment, gelatin has been heated to about 90°C. In some embodiments, the gelatin is added to a concentration of about 90-100 g/L in the final solution. In some embodiments, the gelatin is added to a concentration of about 96 g/L in the final solution. In some embodiments, the time of the reaction between CaCh and Na2CCb is changed from minutes to hours which help to control the reaction and prevent the aggregate formation and recrystallization of vaterite particles. In some embodiments, methods of the present invention include sonification with ultrasound. This modification allows to stabilize the growth of crystals of calcium carbonate and to reduce the size of the resulting cores in the desired range (50-100 nm).
  • the alkaline earth metal carbonate is MgCCb.
  • the alkaline earth metal carbonate is BaCCb.
  • the alkaline earth metal may be any water soluble salt of the alkaline earth metal.
  • the alkaline earth metal salt is a chloride salt.
  • the carbonate source may comprise a water soluble carbonate ion.
  • Examples of such carbonate source include, but are not limited to, soluble carbonates or hydrocarbonates, such as sodium carbonate, sodium hydrocarbonate, ammonium carbonate, ammonium bicarbonate potassium carbonate, or potassium bicarbonate.
  • the carbonate source is sodium hydrocarbonate (NaHCCh).
  • the stabilizer can be any organic and inorganic phosphates, sulfonates, sulphates which contain one and more functional groups.
  • the stabilizer is an organic phosphate.
  • the stabilizer is a phosphoric acid ester.
  • the stabilizer is adenosine triphosphate (ATP).
  • Solvents used in the methods of the present invention may include any mixture of solvents or individual solvents which can dissolve the stabilizer and/or the carbonate source substance.
  • solvents that can be used in the methods of the present invention include water and water miscible solvents such as ethanol, acetone, acetonitrile, dimethyl acetamide (DMA), tetrahydrofuran (THF), dioxane, dimethylsulfoxide (DMSO), and dimethylformamide (DMF).
  • DMA dimethyl acetamide
  • THF tetrahydrofuran
  • DMSO dimethylsulfoxide
  • DMF dimethylformamide
  • Other suitable non-exhaustive examples of water-miscible solvents may be found in Perry's Chemical Engineer's Handbook, Sixth Edition, which has been incorporated herein by reference.
  • the solvent in the first solution comprises water. In some embodiments, the solvent in the first solution comprises ethanol. In some embodiments, the solvent in the first solution is a water and ethanol mixture.
  • the solvent in the second solution comprises water. In some embodiments, the solvent in the second solution comprises ethanol. In some embodiments, the solvent in the second solution is a water and ethanol mixture.
  • water and ethanol When water and ethanol are used as a mixture, they may be mixed at a ratio of about 100:0 to about 50:50 by volume in the final solution. When the ratio is 100:0, the solution does not comprise ethanol. It should also be noted that the ratio of water and ethanol in the second solution need not be the same as the ratio of water and ethanol in the first solution, as long as the ratio of water and ethanol in the final solution is about 100:0 to about 50:50. In some embodiments, water and ethanol in the final solution are at a ratio of about 95:5, about 90: 10, about 85: 15, about 80:20, about 75:25, about 70:30, about 65:35, about 60:40, about 55:45, or about 50:50.
  • the final concentration of the alkaline earth metal salt after mixing is between about 0.01 M to about 5 M.
  • the final concentration of the alkaline earth metal salt after mixing may between about 0.05 M to about 4 M, between about 0.1 M to about 3 M, between about 0.2 M to about 2.8 M, between about 0.5 M to about 2.5 M, between about 0.8 M to about 2.4 M, between about 1 M to about 2.3 M, between about 1.5 M to about 2.2 M, between about 1.5 M to about 2.0 M, between about 1.5 to about 1.9 M, between about 1.6 M to about 2.4 M, between about 1.6 M to about 2.2 M, between about 1.6 M to about 2.0 M, between about 1.6 M to about 1.9 M, between about 1.7 M to about 2.2 M, between about 1.7 M to about 2.1 M, between about 1.7 M to about 2.0 M, between about 1.7 M to about 1.9 M, or between about
  • the final concentration of the alkaline earth metal salt after mixing may be about 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, 2.1 M. 2.2 M, 2.3 M, or 2.4 M.
  • the final concentration of the alkaline earth metal salt after mixing may be about 1.61 M, 1.62 M, 1.63 M, 1.64 M, 1.65 M, 1.66 M, 1.67 M, 1.68 M, 1.69 M, 1.70 M, 1.71 M, 1.72 M, 1.73 M, 1.74 M, 1.75 M, 1.76 M, 1.77 M, 1.78 M, 1.79 M, 1.80 M, 1.81 M, 1.82 M, 1.83 M, 1.84 M, 1.85 M, 1.86 M, 1.87 M, 1.88 M, 1.89 M, 1.90 M, 1.91 M, 1.92 M, 1.93 M, 1.94 M, 1.95 M, 1.96 M, 1.97 M, 1.98 M, 1.99 M, or 2.00 M.
  • the final concentration of the alkaline earth metal salt after mixing is between about 0.1 M to about 3 M. In some embodiments, the final concentration of the alkaline earth metal salt after mixing is between about 1.8 M to about 2 M. In one embodiment, the final concentration of the alkaline earth metal salt after mixing is about 1.875M.
  • the final concentration of the carbonate source after mixing is between about 1 mM to about 50 mM.
  • the final concentration of the carbonate source after mixing may be between about 2 mM to about 48 mM, between about 4 mM to about 45 mM, between about 5 mM to about 40 mM, between about 6 mM to about 40 mM, between about 6 mM to about 35 mM, between about 6 mM to about 25 mM, between about 6 mM to about 15 mM, between about 6 mM to about 10 mM, between about 6.5 mM to about 25 mM, between about 6.5 mM to about 20 mM, between about 6.5 mM to about 15 mM, between about 7 mM to about 15 mM, or between about 7 mM to about 10 mM.
  • the final concentration of the alkaline earth metal salt after mixing may be about 6 mM, 6.5 mM, 6.6
  • the final concentration of the carbonate source is between about 6 mM to 40 mM. In some embodiments, the final concentration of the carbonate source is between about 6 mM to 20 mM. In one embodiment, the final concentration of the carbonate source is about 7.5 mM.
  • the final concentration of the carbonate source after mixing is between about 0.1 mg/ml to about 4 mg/ml.
  • the final concentration of the carbonate source after mixing may be between about 0.2 to about 3.4 mg/ml, between about 0.3 to about 3.4 mg/ml, between about 0.4 to about 3 mg/ml, between about 0.5 to about 1 mg/ml, between about 0.5 to about 0.75 mg/ml, between about 0.5 to about 0.7 mg/ml, between about 0.55 to about 0.75 mg/ml, between about 0.55 to about 0.7 mg/ml, between about 0.6 to about 0.75 mg/ml, between about 0.6 to about 0.7 mg/ml, or between about 0.6 to about 0.65 mg/ml.
  • the final concentration of the alkaline earth metal salt after mixing may be about 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.51 mg/ml, 0.52 mg/ml, 0.53 mg/ml, 0.54 mg/ml, 0.55 mg/ml, 0.56 mg/ml, 0.57 mg/ml, 0.58 mg/ml, 0.59 mg/ml, 0.6 mg/ml, 0.61 mg/ml, 0.62 mg/ml, 0.625 mg/ml, 0.63 mg/ml, 0.64 mg/ml, 0.65 mg/ml, 0.66 mg/ml, 0.67 mg/ml, 0.675 mg/ml, 0.68 mg/ml, 0.69 mg/ml, 0.7 mg/ml, 0.71 mg/ml, 0.72 mg/ml, 0.73 mg/ml, 0.74 mg/ml, 0.75 mg/ml, 0.76 mg/ml, 0.77 mg/ml, 0.78
  • the final concentration of the stabilizer after mixing is between about 0.1 mM to about 50 mM.
  • the final concentration of the stabilizer after mixing may be between about 0.2 mM to about 48 mM, between about 0.4 mM to about 45 mM, between about 0.5 mM to about 40 mM, between about 0.5 mM to about 30 mM, between about 0.5 mM to about 25 mM, between about 0.5 mM to about 20 mM, between about 0.5 mM to about 15 mM, between about 0.5 mM to about 10 mM, between about 0.8 mM to about 30 mM, between about 0.8 mM to about 20 mM, between about 0.8 mM to about 10 mM, between about 1 mM to about 40 mM, between about 1 mM to about 35 mM, between about 1 mM to about 25 mM, between about 1 mM to about 15 mM, between about
  • the final concentration of the alkaline earth metal salt after mixing may be about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.25 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.2 mM, 2.5 mM, 2.8 mM, 3 mM, 3.2 mM, 3.5 mM, 3.8 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM, 10 mM, 15
  • the final concentration of the stabilizer is between about 1 mM to 40 mM. In some embodiments, the final concentration of the stabilizer is between about 1 mM to 5 mM. In one embodiment, the final concentration of the stabilizer is about 1.25 mM.
  • the final concentration of the stabilizer after mixing is between about 0.1 mg/ml to about 4 mg/ml.
  • the final concentration of the stabilizer after mixing may be between about 0.2 to about 3.4 mg/ml, between about 0.3 to about 3.4 mg/ml, between about 0.4 to about 3 mg/ml, between about 0.5 to about 1 mg/ml, between about 0.5 to about 0.75 mg/ml, between about 0.5 to about 0.7 mg/ml, between about 0.55 to about 0.75 mg/ml, between about 0.55 to about 0.7 mg/ml, between about 0.6 to about 0.75 mg/ml, between about 0.6 to about 0.7 mg/ml, or between about 0.6 to about 0.65 mg/ml.
  • the final concentration of the alkaline earth metal salt after mixing may be about 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.51 mg/ml, 0.52 mg/ml, 0.53 mg/ml, 0.54 mg/ml, 0.55 mg/ml, 0.56 mg/ml, 0.57 mg/ml, 0.58 mg/ml, 0.59 mg/ml, 0.6 mg/ml, 0.61 mg/ml, 0.62 mg/ml, 0.625 mg/ml, 0.63 mg/ml, 0.64 mg/ml, 0.65 mg/ml, 0.66 mg/ml, 0.67 mg/ml, 0.675 mg/ml, 0.68 mg/ml, 0.69 mg/ml, 0.7 mg/ml, 0.71 mg/ml, 0.72 mg/ml, 0.73 mg/ml, 0.74 mg/ml, 0.75 mg/ml, 0.76 mg/ml, 0.77 mg/ml, 0.78
  • the molar concentration of stabilizer and carbonate source in the final concentration has a ratio from 1:1 to 1:6.
  • the amount of carbonate source in the final solution may be about one, two, three, four, five or six times (including all integers and decimal points in between, e.g., 1.5, 1.6, 1.7. 1.8, etc.) of the amount of the stabilizer.
  • the method of preparing an alkaline earth metal carbonate nanoparticle may include a step for stabilizing the nanoparticle.
  • the alkaline earth metal carbonate nanoparticle may be resuspended in a stabilization solution.
  • the stabilization solution may comprise any type of phosphoric acid salts and their derivatives (e.g. organo esters).
  • the stabilization solution is a phosphate solution.
  • the stabilization solution comprises sodium triphosphate.
  • the pH of salt solution is not less than 6.
  • the sodium triphosphate is at a concentration between about 1 mg/ml to about 30 mg/ml.
  • the sodium triphosphate may be at a concentration of between about 1 mg/ml to 25 mg/ml, about 1 mg/ml to 20 mg/ml, between about 2 mg/ml to 20 mg/ml, between about 2 mg/ml to 15 mg/ml, between about 4 mg/ml to 18 mg/ml, between about 5 mg/ml to 12 mg/ml, between about 5 mg/ml to 10 mg/ml, between about 5 mg/ml to 9 mg/ml, between about 5 mg/ml to 8 mg/ml, between about 6 mg/ml to 10 mg/ml, between about 6 mg/ml to 9 mg/ml, or between about 6 mg/ml to 8 mg/ml.
  • the sodium triphosphate may be at a concentration of about 3 mg/ml, 3.5 mg/ml, 4 mg/ml, 4.5 mg/ml, 5 mg/ml, 5.5 mg/ml, 6 mg/ml, 6.5 mg/ml, 7 mg/ml, 7.5 mg/ml, 8 mg/ml, 8.5 mg/ml, 9 mg/ml, 9.5 mg/ml, 10 mg/ml, 10.5 mg/ml, 11 mg/ml, 11.5 mg/ml, 12 mg/ml, 12.5 mg/ml, 13 mg/ml, 13.5 mg/ml, 14 mg/ml, 14.5 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, or 20 mg/ml.
  • the sodium triphosphate is at a concentration of about 7 mg/ml.
  • the method of preparing an alkaline earth metal carbonate nanoparticle may also include one or more wash steps.
  • the wash step(s) may be carried out before, after and/or during the stabilizing step.
  • the alkaline earth metal carbonate nanoparticle may be washed by any solution that is capable of removing any excess reagents or impurity while preserving the integrity of the nanoparticle.
  • Such solution may include without limitation, water (e.g., hot water of about 70°C), and ethanol solution, e.g., up to 70% ethanol solution. Washing may be done once, twice, three times, four times, five times or more (e.g., 10, 20 times etc.).
  • the alkaline earth metal carbonate nanoparticle prepared using the method of the present disclosure may be resuspended and/or stored in a storage solution to increase long-time stability of the particles.
  • a storage solution may be a water free solution.
  • the phosphate solution is an ethanol solution.
  • the concentration of sodium triphosphate is between about 0.01 mg/ml to about 2 mg/ml.
  • the concentration of sodium triphosphate is between about 0.01 mg/ml to about 1.5 mg/ml, between about 0.01 mg/ml to about 1 mg/ml, between about 0.02 mg/ml to about 1 mg/ml, between about 0.02 mg/ml to about 0.8 mg/ml, between about 0.05 mg/ml to about 0.5 mg/ml, between about 0.1 mg/ml to about 0.5 mg/ml, between about 0.1 mg/ml to about 0.4 mg/ml, between about 0.2 mg/ml to about 0.5 mg/ml, or between about 0.2 mg/ml to about 0.4 mg/ml.
  • the concentration of sodium triphosphate is about 0.02 mg/ml, 0.05 mg/ml, 0.08 mg/ml, 0.1 mg/ml, 0.15 mg/ml, 0.2 mg/ml, 0.21 mg/ml, 0.22 mg/ml, 0.23 mg/ml, 0.24 mg/ml, 0.25 mg/ml, 0.26 mg/ml, 0.27 mg/ml, 0.28 mg/ml, 0.29 mg/ml, 0.3 mg/ml, 0.31 mg/ml, 0.32 mg/ml, 0.33 mg/ml, 0.34 mg/ml, 0.35 mg/ml, 0.4 mg/ml, 0.45 mg/ml, 0.5 mg/ml, 0.55 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml
  • calcium carbonate nanoparticles are stabilized with the addition of stabilizing agents, such as adenosine triphosphate and other molecules (Qi C, Zhu YJ, Lu BQ, Zhao XY, Zhao J, Chen F, Wu J. Small. 2014 May 28;10(10):2047-56, which is herein incorporated by reference in its entirety) which bind to the surface of the core of capsules, preventing it from the dissolution and recrystallization. It is not a routine optimization because smaller particles have less aggregative stability by comparing with microparticles analog.
  • stabilizing agents such as adenosine triphosphate and other molecules (Qi C, Zhu YJ, Lu BQ, Zhao XY, Zhao J, Chen F, Wu J. Small. 2014 May 28;10(10):2047-56, which is herein incorporated by reference in its entirety) which bind to the surface of the core of capsules, preventing it from the dissolution and recrystallization. It is not a routine optimization because smaller particles have
  • the present invention also provides an alkaline earth metal carbonate nanoparticle produced by the method described herein.
  • the alkaline earth metal carbonate nanoparticle produced according to the present invention may have a diameter of about 50 nm to about 300 nm.
  • the alkaline earth metal carbonate nanoparticle may have a diameter of about 50 nm to about 70 nm, about 50 nm to about 80 nm, about 50 nm to about 90 nm, about 50 nm to about 100 nm, about 50 nm to about 120 nm, about 50 nm to about 150 nm, about 50 nm to about 200 nm, about 60 nm to about 120 nm, about 60 nm to about 150 nm, about 80 nm to about 150 nm, about 80 nm to about 200 nm, or about 80 nm to about 250 nm.
  • the alkaline earth metal carbonate nanoparticle may have a diameter of about 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, or 300 nm.
  • the alkaline earth metal carbonate nanoparticle has a diameter of about 50 nm to about 150 nm. In some embodiments, the alkaline earth metal carbonate nanoparticle has a diameter of about 50 nm to about 100 nm.
  • the present invention also provides a method of preparing a nanocapsule having a diameter of about 50 nm to about 500 nm comprising: a) coating an alkaline earth metal carbonate nanoparticle having a diameter of about 50 nm to about 150 nm with one or more shell layers, each shell layer comprising at least one biodegradable material comprising two or more electronegative and/or electropositive groups, and optionally adsorbing a cargo to the one or more shell layers; and b) optionally, removing the alkaline earth metal carbonate nanoparticle.
  • the alkaline earth metal carbonate nanoparticle used in step (a) is produced by a method described herein.
  • Methods for coating an alkaline earth metal carbonate nanoparticle with one or more shell layers may include those described in the art and in the Examples section below, for example, the layer-by-layer (LbL) technique.
  • LbL layer-by-layer
  • successive and alternating anionic and cationic layers are coated onto the core.
  • the one or more shell layers are added without a washing step between the addition of layers.
  • Removal of the alkaline earth metal carbonate nanoparticle may be achieved by, for example, contacting the alkaline earth metal carbonate nanoparticle with an acid or sodium EDTA solution.
  • the method of preparing a nanocapsule may include a sorting step to ensure that the nanocapsules have a homogenous distribution of size.
  • the nanocapsules may be passed through one or more filters with appropriate size or a microfluidic channel system.
  • Materials used to form the shell layer may comprise more than one electronegative and/or electropositive group. Other preferred properties of the shell materials include the presence of two or more solvent ionized functional groups (including pH-ionizing), low local and system toxicity, and biodegradation possibility or eliminating a substance from the body unchanged.
  • Electronegative groups useful in the present invention include, but are not limited to, sulfonic acid, sulfuric acid, carboxylic acid, phosphoric acid, polyphosphoric acids, and phenolic hydroxyl, and combinations thereof.
  • Electropositive groups useful in the present invention include, but are not limited to, primary amines or their onium cations, secondary amines or their onium cations, tertiary amines or their onium cations, quaternary ammonium compounds, and cyclic amines or their onium cations, and combinations thereof.
  • the biodegradable material comprising two or more electronegative groups is selected from dextran sulfate, poly(4-vinylphenol), poly(sodium 4- styrenesulfonate), fucoidan, polyethylene glycol, bovine serum albumin, and tannic acid.
  • the biodegradable material comprising two or more electropositive groups is selected from polyacrylamide, poly-l-lysine, poly-l-arginine, poly(allylamine hydrochloride), poly-l-ornithine, and chitosan.
  • the present invention provides a nanocapsule produced by the method described herein.
  • the present invention provides a nanocapsule having a diameter of 50 nm to 500 nm comprising: a) a core comprising an alkaline earth metal carbonate nanoparticle; b) one or more shell layers coating the core comprising at least one biodegradable material comprising two or more electronegative and/or electropositive groups; and c) a cargo.
  • the alkaline earth metal carbonate is selected from CaCCb, MgCCb, and BaCCb. In some embodiments, the alkaline earth metal carbonate is CaCCb. In some embodiments, the CaCCb particle is selected from a vaterite particle, a calcite particle, an aragonite particle, and a particle of amorphous CaCCb. [00147] In some embodiments, the CaCCb particle is an amorphous CaCCb particle.
  • the alkaline earth metal carbonate is MgCCb.
  • the alkaline earth metal carbonate is BaCCb.
  • the present invention provides a hollow nanocapsule having a diameter of 50 nm to 500 nm comprising: a) one or more shell layers comprising at least one biodegradable material comprising two or more electronegative and/or electropositive groups, and b) a cargo.
  • the hollow nanocapsule may be produced by first using a core onto which the one or more shell layers of the biodegradable material(s) are coated. After the biodegradable material(s) have been coated and/or the cargo has been loaded onto the particle, the core can be removed.
  • a low nanosized alkaline earth metal carbonate (e.g., CaCCb) nanoparticle is used as the core.
  • the core is removed at the end of the process, the center of the capsule is void.
  • a nanocapsule of the present invention may have a diameter of about 50 nm to about 500 nm, depending on the size of the alkaline earth metal carbonate nanoparticle core.
  • the nanocapsule may have a diameter of about 50 nm to about 400 nm, about 50 nm to about 300 nm, about 50 nm to about 250 nm, about 50 nm to about 200 nm, about 50 nm to about 150 nm, about 80 nm to about 400 nm, about 80 nm to about 300 nm, about 80 nm to about 250 nm, about 80 nm to about 200 nm, about 80 nm to about 150 nm, about 80 nm to about 300 nm, about 100 nm to about 400 nm, about 100 nm to about 300 nm, about 100 nm to about 250 nm, about 100 nm to about 200 nm, about 100 nm to about 150 nm, about 120 nm to
  • a nanocapsule of the present invention has a diameter of about 50 nm to about 300 nm. In some embodiments, a nanocapsule of the present invention has a diameter of about 100 nm to about 200 nm.
  • the biodegradable material in the layers may be the same or different. It is possible to include 2, 3 or even more shell layers which may have different size. Similarly, the cargo can be the same or different in each layer.
  • Biodegradable materials that can be used in preparing a nanocapsule of the present invention include, but are not limited to, an oligopeptide, a polypeptide, an oligosaccharide, a polysaccharide, a glycopeptide, a glycolipid, a natural nucleic acid, an artificial nucleic acid, a polyphenol, a synthetic biodegradable polymer, and a biodegradable cross-linking agent.
  • Such biodegradable material may comprise two or more ionizable functional groups.
  • biodegradable materials used in preparing a nanocapsule of the present invention include natural and artificial oligopeptides and polypeptides with functional groups which that can be ionized by dissociation or acid protonation.
  • positively charged polymers may include oligopeptides or polypeptides comprising primarily basic amino acids such as arginine, ornithine, lysine, histidine including their acidic salts.
  • negative charged polymers may include oligopeptides or polypeptides comprising primarily acidic amino acids as such as aspartic acid and glutamic acid.
  • biodegradable materials used in preparing a nanocapsule of the present invention include an oligosaccharide or a polysaccharide with functional groups which can be ionized by dissociation or acid protonation.
  • oligosaccharide or polysaccharide may comprise a monomer such as glucose, fructose, ribose, idose, allose, altrose, gulose, talose, fucose, mannose, galactose, neuraminic acid, and glucuronic acids, and combinations thereof.
  • biodegradable materials used in preparing a nanocapsule of the present invention include glycopeptides and glycolipids that have the properties described above.
  • biodegradable materials used in preparing a nanocapsule of the present invention include natural and artificial nucleic acids. Natural and artificial nucleic acids may be used as a negative charged polyelectrolyte layer.
  • biodegradable materials used in preparing a nanocapsule of the present invention include polyphenols such as those from the tannin class.
  • biodegradable materials used in preparing a nanocapsule of the present invention include a synthetic biodegradable polymer such as a polyester, a polyamine, polydiacetylene (PDA), polyethylene glycol (PEG), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), polyvinyl alcohol (PVA), polyhydroxybutyrate (PHB), polyvinylpyrrolidone (PVP), and derivatives and co-polymers thereof.
  • a synthetic biodegradable polymer such as a polyester, a polyamine, polydiacetylene (PDA), polyethylene glycol (PEG), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), polyvinyl alcohol (PVA), polyhydroxybutyrate (PHB), polyvinylpyrrolidone (PVP), and derivatives and co-polymers thereof.
  • PDA polydi
  • biodegradable materials used in preparing a nanocapsule of the present invention include low molecular substances which contain more than one functional groups that can be ionized by dissociation or acid protonation.
  • such low molecular substances may be phospholipids, biotin, cholic acids, or folic acid.
  • biodegradable materials used in preparing a nanocapsule of the present invention include a cross-linking agent such as glutaraldehyde.
  • the cargo to be delivered using the nanoparticles and/or nanocapsules of the invention include, but not limited to, nucleic acids, peptides, proteins, or small molecules.
  • Nucleic acids cargos may include, but not limited to, a single-stranded DNA, a double- stranded DNA (e.g., plasmid DNA), a mini circle DNA, an RNA, and synthetic analogs and derivatives thereof.
  • RNA cargos include interfering RNA (RNAi) molecules (e.g., siRNA molecule or a shRNA molecule), dsRNA, RNA polymerase III transcribed RNAs, messenger RNA (mRNA), and antisense nucleic acids.
  • Nucleic acids can have a native form or a conjugate form with polymer structures or lipids, or a salt form.
  • biologicaly active molecules delivered in the form of nucleic acids include: genome editing tools such as CRISPR proteins (e.g. Cas9, Cpfl), TALEN, ZFNs, functional and immunogenic peptides, antibodies, chimeric antibodies (e.g. bispecific antibodies) and polypeptides derived from antibodies, such as, for example, single chain variable fragments (scFv), Fab, Fab', F(ab')2, and Fv fragments, polypeptides derived from T Cell receptors, such as, for example, TCR variable domains, secreted factors (e.g., cytokines, growth factors, including synthetic and recombinant factors), enzymes (e.g. transposase), receptors, chimeric antigen receptors (CAR).
  • genome editing tools such as CRISPR proteins (e.g. Cas9, Cpfl), TALEN, ZFNs, functional and immunogenic peptides, antibodies, chimeric antibodies (e.g. bispecific antibodies) and polypeptides
  • Protein-nucleic acid complexes can also be delivered using the nanoparticles and/or nanocapsules of the invention.
  • a protein-nucleic acid complex may be a protein-nucleic acid complex associated with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/nuclease gene system, such as gRNA-Cas9 or gRNA-Cpfl .
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the guide RNA (gRNA) and the CRISPR protein e.g., Cas9, Cpfl
  • Cas9, Cpfl may be loaded simultaneously or sequentially.
  • the CRISPR protein (e.g., Cas9, Cpfl) and the gRNA may be loaded in two sets of nanocapsules.
  • the populations of the two sets of nanocapsules may be mixed in a ratio of approximately 1 : 1 and this mixture is used for transferring/transfecting the target cells.
  • the gRNA and the CRISPR protein (e.g., Cas9, Cpfl) may be pre-complexed to form a ribonucleoprotein which is then loaded onto the nanocapsules. By doing this, the gRNA can be protected in the ribonucleoprotein complex which reduces the possibility of nucleic acid degradation.
  • the CRISPR protein (e.g., Cas9, Cpfl) may be loaded as an mRNA encoding the protein, or in a vector which encodes both the CRISPR protein and the gRNA.
  • Peptides or proteins that can be delivered using the nanoparticles and/or nanocapsules of the invention can include peptides (e.g.
  • antibodies polypeptides derived from antibodies, such as, for example, single chain variable fragments (scFv), Fab, Fab', F(ab')2, and Fv fragments, polypeptides derived from T Cell receptors, such as, for example, TCR variable domains, secreted factors (e.g., cytokines, growth factors).
  • scFv single chain variable fragments
  • Fab fragment antigen-binding protein
  • Fab' fragment antigen binding domains
  • F(ab')2 fragment antigen binding
  • Fv fragments polypeptides derived from T Cell receptors
  • TCR variable domains such as, for example, TCR variable domains
  • secreted factors e.g., cytokines, growth factors.
  • peptides or a proteins include a CRISPR protein (e.g., Cas9, Cpfl), a zinc finger nuclease (ZFN), and a transcription activator-like effector nuclease (TALEN).
  • Small molecules that can be delivered using the nanoparticles and/or nanocapsules of the invention include, but not limited to, an antineoplastic drug (such as doxorubicin, vinblastin, etoposide, cisplatin, monomethyl auristatin E, and ozogamycin), an immunosuppressant (such as tacrolimus, sirolimus, ciclosporin, and fmgolimod), a tyrosine kinase inhibitor (such as dasatinib, ibrutinib, ruxolitinib, tofacitinib, axitinib, sorafenib, idelalisib, venetoclax, and erlotinib), a demethylating agent (such as decitabine), a histone deacetylase (HDAC) inhibitor (such as vorinostat), an indoleamine 2, 3 -di oxygenase (IDO) inhibitor (such as
  • AIM2, cGAS, STING and TLR pathway antagonists including but not limited to N-(4-Ethylphenyl)-N’-lH-indol-3-yl-urea (H-151) and N-(4-iodophenyl)-5-nitro-2- furancarboxamide (C-176)).
  • any of the following may be utilized as a carrier for a cargo: 1) an alkaline earth metal carbonate nanoparticle described herein; 2) a nanocapsule described herein having a diameter of 50 nm to 500 nm comprising: a) a core comprising an alkaline earth metal carbonate nanoparticle, and b) one or more shell layers coating the core comprising at least one biodegradable material comprising two or more electronegative and/or electropositive groups; and 3) a hollow nanocapsule described herein having a diameter of 50 nm to 500 nm comprising one or more shell layers comprising at least one biodegradable material comprising two or more electronegative and/or electropositive groups.
  • Cargo loading may be achieved in several ways.
  • cargos can be internalized into the nanoparticle.
  • Cargos may be co-precipitated with the nanoparticle, for example, by adding cargo to the first solution comprising a carbonate source, a stabilizer, and a solvent. Co precipitation may be performed as described, for example, in Brodskaia A.V. et ah, Antiviral Res. 2018 Oct;158:147-160, which is incorporated herein by reference in its entirety for all purposes.
  • Cargos may also be adsorbed onto a preobtained nanoparticle before shell coating.
  • Adsorption may be achieved by contacting the nanoparticle with any compatible cargo solution, or by freezing-induced loading method [German SV et al High-efficiency freezing-induced loading of inorganic nanoparticles and proteins into micron- and submicron-sized porous particles Scientific Reports volume 8, Article number: 17763 (2016)] which leads to cargo adsorption onto the nanoparticle structure under the influence of gradient crystallization front.
  • the cargo may be liberated after the whole shell has been dissolved.
  • the cargo may be encapsulated by the nanocapsule shell once the core is removed.
  • Cargos may also be adsorbed onto one or more shell layers.
  • nucleic acids may be adsorbed onto positively charged polyelectrolyte layer.
  • cargos may be co-precipitated with a component of one or more shell layers and thus remain embedded in the one or more shell layers.
  • the nanocapsules of the present invention may be further comprise one or more of the following agents: magnetic particles, fluorescent dyes, sensors, radionucleids, microreactors and/or specific ligands which bind to receptors of the target cells. Possible ways of encapsulating such agents in a nanocapsule are shown in Figure 9.
  • a nanocapsule of the present invention may be coupled with a specific ligand which bind to receptors of the target cells to improve the targeting specificity of the nanocapsules. It is also desirable to include specific compounds which increase the efficiency of uptake into certain cells. This may be in particular advantageous when the nanocapsules are applied to patients, for example by injection.
  • the present invention provides a preparation of alkaline earth metal carbonate nanoparticles produced by the method described herein.
  • the preparation may comprise at least 50% of the nanoparticles having a diameter of 50 nm to 300 nm.
  • the composition may comprise at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, about 50%, about 55%, about 60%, about 65%, about 70%, about 72%, about 74%, about 76%, about 78%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or higher of the nanoparticles having a diameter of 50 nm to 300 nm.
  • at least 90% of the nanoparticles in the preparation have a diameter of about 50 nm to about 300 nm.
  • the preparation may comprise at least 50% of the nanoparticles having a diameter of 50 nm to 150 nm.
  • the preparation may comprise at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, about 50%, about 55%, about 60%, about 65%, about 70%, about 72%, about 74%, about 76%, about 78%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or higher of the nanoparticles having a diameter of 50 nm to 150 nm.
  • the present invention provides a composition comprising a plurality of the nanoparticles and/or nanocapsules described herein and a pharmaceutically acceptable carrier.
  • the composition may comprise at least 50% of the nanocapsules having a diameter of 100 nm to 200 nm.
  • the composition may comprise at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, about 50%, about 55%, about 60%, about 65%, about 70%, about 72%, about 74%, about 76%, about 78%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or higher of the nanocapsules having a diameter of 100 nm to 200 nm.
  • the composition comprises at least 90% of the nanocapsules having a diameter of 100 nm to 200 nm.
  • the composition may comprise at least 50% of the nanocapsules having a diameter of 100 nm to 200 nm.
  • the composition may comprise at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, about 50%, about 55%, about 60%, about 65%, about 70%, about 72%, about 74%, about 76%, about 78%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or higher of the nanocapsules having a diameter of 100 nm to 200 nm.
  • the composition comprises at least 90% of the nanocapsules having a diameter of
  • compositions comprising nanoparticles and/or nanocapsules disclosed herein may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins polypeptides or amino acids such as glycine
  • antioxidants e.g., chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • Compositions may further comprise one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • An injectable pharmaceutical composition is preferably sterile.
  • the present invention provides a method of delivering a cargo to a target cell of a subject comprising administering to the subject an effective amount of the nanoparticles and/or nanocapsules described herein, or a composition thereof.
  • the target cell is a mammalian cell. It is contemplated that the nanoparticles and/or nanocapsules described herein, or a composition thereof, may be suitable for transfecting any kind of target cells in mammalian organism. Exemplary types of target cells may include mammalian primary stem cells, cells forming stem-cells niches and differentiated cells, tumor cells (including primary tumor cells) and cells forming tumor microenvironment.
  • Stem cells that may be targeted for cargo delivery include, but are not limited to, hematopoietic stem cells and their progeny, induced pluripotent stem cells, embryonic stem cells, a retinal stem cell, egg cells, spermatozoons, germ layers cells and their progeny (e.g., endoderm, mesoderm, ectoderm, neural crest).
  • progeny e.g., endoderm, mesoderm, ectoderm, neural crest.
  • target cells include a T-cell, a B-cell, an NK-cell, a monocyte, a macrophage, a fibroblast, a neuron (including photoreceptors), an epithelial cell, a light-sensing cell, a leukocyte or a progenitor thereof, an erythrocyte or a progenitor thereof, and a hepatocyte.
  • Epithelial cells that may be targeted for cargo delivery include, but are not limited to, an intestinal epithelium cell, a retinal pigmented epithelial (RPE) cell, an esophageal epithelial cell, a glandular epithelial cell, a bladder epithelium cell, a prostate epithelial cell, a mesothelium cell, an epidermis cell, an endothelium cell, an alveolar epithelium cell, a germinal epithelium cell, a mucosal epithelium cell, a conjunctival epithelium cell, a glomerular epithelium, a respiratory epithelium cell, and an epithelial stem cell.
  • RPE retinal pigmented epithelial
  • routes of administration suitable for the present invention may include enteral and gastrointestinal routes, including, but not limited to, taken by mouth (orally), placed under the tongue (sublingually) or between the gums and cheek (buccally), inserted in the rectum (rectally) or vagina (vaginally), or direct enteral administration (the gastric tube of via gastrostomy, duodenal tube).
  • enteral and gastrointestinal routes including, but not limited to, taken by mouth (orally), placed under the tongue (sublingually) or between the gums and cheek (buccally), inserted in the rectum (rectally) or vagina (vaginally), or direct enteral administration (the gastric tube of via gastrostomy, duodenal tube).
  • Parenteral and topical administration may also be used, which includes, but not limited to, epicutaneous (application onto the skin), transdermal (i.e.
  • transdermal patch intravenous, intra-arterial, intra- articular, intracardiac, intracavernous, or intravaginal injections and infusions, intratesticular, intradermal (into the skin itself), intralesional, subcutaneous, hypodermic injections and infusions, intramuscular, intraosseous injections and infusions, intraocular, subconjunctival, intraperitoneal (infusion or injection into the peritoneum), intrathecal (into the spinal canal), intrauterine, intravesical infusion (urinary bladder), intravitreal, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), perivascular injections and infusions, transmucosal (any mucous membrane), nasal administration, inhalation, nebulization, placed in the eye (such as eyedrops) or the ear (by the otic route), epidural (peridural) (injection or infusion into the epidural space), extra-amn
  • the subject is an experimental or veterinary animal such as mice, rats, rabbits, dogs, cats, pigs, horses, cattle, and primates.
  • the subject is human.
  • Submicron vaterite particles Submicron particles of CaCCh were prepared as described by [Parakhonskiy B.V., et. al Tailored intracellular delivery via a crystal phase transition in 400 nm vaterite particles. Biomater. Sci., 2013,1, 1273-1281] with additional modification. Briefly, two salts solutions (CaCb and NaCCh) were diluted either in ethylene glycol solution or glycerol solution. Next, the obtained solutions were mixed at the 900rpm magnetic field for 3 hours. For optimization of synthesis time, ultrasonic sonication instead of magnetic mixing was applied. Also, gelatin was added for reducing calcium carbonate particles size. To prevent recrystallization, the samples were dehydrated with ethanol and dried afterward.
  • Nanosized vaterite particles Certain conditions were tested and modified to obtain nanoscale particles of vaterite with the smallest average diameter to synthesize a template for the assembly of biocompatible polymeric capsules.
  • Gelatin from porcine skin (4.8g) was dissolved in 50ml distilled water (dFhO) and was heated to 90°C.
  • Na2CCh and CaCh salts solutions (0.1M concentration) were diluted in 20ml 87% glycerol and were added in the heated gelatin solution. The total solution was rapidly mixed under 500rpm magnetic stirring and sonicated during lOmin by Ultrasonic Processor at a frequency of 20 kHz and radiation power of 125W.
  • the synthesized CaCCh particles were washed twice by hot water (70°C) and were kept in chymotrypsin solution during 1 hour for total clearing of vaterite particles from gelatin.
  • Nanosized amorphous calcium carbonate particles were tested.
  • commercial adenosine triphosphate (ATP) was added.
  • ATP contains sodium carbonate and sodium hydrocarbonate at concentrations of 10 mg/ml and 20 mg/ml respectively in water/ethanol 50/50. Due to this, stabilized calcium carbonate particles with a size of 50-100nm was obtained at mixing of calcium chloride (120m1) in water/ethanol 50/50.
  • this optimized protocol allows to produce nanosized (50nm) amorphous calcium carbonate particles at large volumes, excluding the using of ultrasound and gelatin. Size and morphology of nanoparticles was confirmed by DLS and SEM.
  • the size distribution was measured using a zetasizer Dynamic light scattering (DLS). For controlling of CaCCh crystallization Transmission Raman Spectroscopy was applied.
  • a X-ray diffractometer on finely powdered samples at scanning speed of 0.3° 20 s _1 , l 0.154 nm with Cu Ka as a radiation source (44.8 kV and 0.65 mA) was used for identification of the prepared calcium carbonates polymorphs.
  • Statistical image analysis was performed using Image!
  • pOptiVECTM-TOPO vector (Invitrogen, Life Technologies) with inserted eGFP gene was used to express the green fluorescence protein (GFP) in a cell line as a transfection marker.
  • Plasmid DNA was obtained from E.coli DH5alpha, extraction was performed using PureLinkTM HiPure Plasmid DNA Purification Kit (Invitrogen, Life Technologies). Concentration of plasmid DNA was evaluated by spectroscopy (NanoDrop®ND-1000, ThermoScientific). T7 promoter and eGFP gene was cloned into pJET (CloneJET PCR Cloning Kit, Thermo Scientific) for the purpose of in vitro transcription.
  • mRNA was synthesized using T7 mScriptTM Standard mRNA Production System (CellScript) and included 5 '-capping and polyadenylation (poly(A)). PJet 1.2 with eGFP gene linearized by Hindlll was used as a template for synthesis. eGFP-encoding mRNA were synthesized without modified nucleosides. After poly(A)-tailing and caping reactions, the mRNA was cleaned up by DNAse. Quality of RNA were assessed by agarose gel electrophoresis, concentration was evaluated spectroscopically . mRNA was stored frozen in -80°C and subjected to minimal freeze-thaw cycles.
  • RNA/DNA was performed using the layer-by-layer (LbL) technique.
  • LbL technique is based on the sequential adsorption of oppositely charged molecules, such as poly electrolytes, onto a charged sacrificial template.
  • the biocompatible polyelectrolytes was used: Dextran Sulfate (DEXS) and Poly-L-arginine hydrochloride (PARG) in concentration 1 mg/ml for each layer.
  • BSA FITC was used as the zero layer and loaded into the core by the freezing-induced loading method (German, S. V., Sci. Rep.
  • T-cells were cultured in complete RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1.5 gL _1 sodium bicarbonate, 4.5 gL _1 glucose, 10 mM HEPES, 1.0 mM sodium pyruvate and 0.05 mM 2-mercaptoethanol.
  • FBS heat-inactivated fetal bovine serum
  • 2 mM L-glutamine 1.5 gL _1 sodium bicarbonate
  • 4.5 gL _1 glucose 10 mM HEPES
  • 1.0 mM sodium pyruvate 0.05 mM 2-mercaptoethanol.
  • the immunophenotype of cells used in further experiments were confirmed by flow cytometry using anti-CD3/anti-CD45 antibodies (BD Bioscience, USA).
  • Viability assay For evaluation of cytotoxicity of nanocapsules, AlamarBlue cell viability assay (Thermo Fisher Scientific) was performed. After 24 hours of incubation with nanocapsules cells were washed twice with PBS, 0.01% AlamarBlue solution in PBS was added and cells were incubated for 1 h at 37 °C. Cells were then seeded using microspin centrifuge FV-2400 and supernatants were collected. Absorbance was measured at 535 nm/600 nm 247 wavelengths.
  • Example 1 CaCCh nanoparticles fabrication at various synthesis conditions [00214] For the preparation of carbonate particles, several syntheses have been considered and sizes of CaCCh crystals obtained by different protocols were compared.
  • Nanosized vaterite preparation and morphology characterization Certain conditions were tested and modified to obtain nanosized vaterite particles with the lowest average size value of about 50 nm for biocompatible polymeric capsules synthesis.
  • gelatin 4.8 g
  • Salts solutions of Na2CC and CaCb (at a concentration of 0.1 M) diluted in 20 ml of 87% glycerol were added to the heated gelatin solution.
  • the total solution was stirred at 500 rpm with magnetic stirring and was sonicated for 10 minutes with an ultrasonic processor with a frequency of 20 kHz and a radiation power of 125 W.
  • the synthesized CaCCh particles were washed twice with hot water (70 °C) and incubated in chymotrypsin solution for 1 hour to completely clear the obtained particles from gelatin.
  • the crystals of calcium carbonate initially sedimented as an amorphous precipitate of spherical granules with a diameter from 10 nm to 70 nm.
  • Subsequent processes of transformation and dissolution-recrystallization lead to the formation of a mixture of crystalline forms of calcium carbonate (hexahydrate and monohydrate) and three anhydrous crystalline polymorphic forms (vaterite, aragonite and calcite).
  • the task is to determine the crystal structure of vaterite calcium carbonate.
  • FIG. 3A illustrates the XRD patterns of the prepared CaCCh. The peaks at 38.5°, 38.7°, 53.1°, 56.9°, 63.4°, 71.0° and 76.9° 2Q are attributed to the formation of aragonite (Johan P. R. De V Amsterdam. Am. Mineral. 1971, 56 (5-6), 758-767), the peaks 136.0° and 81.6° 2Q are attributed to the formation of calcite (Sitepu, H. Powder Diffr.
  • XRD X-ray diffraction
  • Raman spectroscopy Raman spectroscopy
  • TEM transmission electron microscopy
  • DLS dynamic light scattering
  • Example 3 Polymeric nanocapsules preparation and DNA/RNA loading
  • biocompatible polyelectrolytes dextran sulfate (DEXS) and poly-L- arginine hydrochloride (PARG) were used at concentrations of 1 mg/ml.
  • a new method has been developed for encapsulating mRNA.
  • BSA FITC was used as the zero layer and loaded into the core by the freezing-induced loading method for nanocapsules visualization. 1-10 pg of mRNA was diluted in 50 pi RNAse-free ddFEO. Then, the mRNA was coated with a PARG layer, thereby forming the PARG + mRNA complex, which would preserve structure and integrity of mRNA inside the nanocapsule.
  • Solution A contained a stabilizer - 20 m ⁇ of ATP in water (10 mg/ml), a carbonate source - 20 m ⁇ of sodium hydrocarbonate in water (10 mg/ml), and a solvent - 60 m ⁇ of water and 100 m ⁇ of ethanol. After addition of all compounds, the solution was stirred by pipetting.
  • Solution B contained 5M CaCb in ethanol/water mixture (50% / 50% by volume). [00222] 120 m ⁇ of Solution B was added to all volume of Solution A. Resulting solution was stirred by pipetting. After this all particles were centrifuged out for 2 minutes at 12,400g. Extracted particles were gently washed by water twice without resuspension of residue. Obtained calcium carbonate/ ATP hybrid particles was resuspended in sodium triphospate solution (7 mg/ml) to increase stability of these particles ( Figure 5). The particles were then washed once by water and resuspended in 500 ml of 0.28 mg/ml sodium triphospate solution.
  • Coating nanoparticles with polyelectrolyte layers can be achieved by various methods. Two exemplary methods are described below.
  • polyelectrolyte layers are added without washing using the following steps:
  • First step addition of poly cation with functional group at a concentration of 5 mM or more in 0.1M MES buffer titrated by sodium hydroxide to a pH of about 6.5;
  • Second step addition of polyanion with functional group at a concentration of 10 mM or more in 0.1M MES buffer titrated by sodium hydroxide to a pH of about 6.5; and [00227] Third step: addition of poly cation with functional group at a concentration of 5 mM or more in 0.1M MES buffer titrated by sodium hydroxide to a pH of about 6.5.
  • Figure 8A As shown in Figure 8A, after 24 hours of incubation, there was no significant impact of nanocapsules uptake on the viability of cells.
  • Figure 8B represents flow cytometry data plots, reflecting cellular uptake. A dose-dependent increase was observed in cell-nanocapsule association up to almost 100% in 10 pg.
  • Flow cytometry plots show profiles of separated human T-cells treated with nanocapsules labeled by FITC at different concentrations (0.15-25 pg). Dot-plot of cells forward light scatter (FSC) and side light scatters (SSC) is reflected in Figure 8A. The cellular uptake efficiency of nanocapsules was about 99% for 25 pg. Nanocapsules at same concentrations, as described above, have not significantly impaired cell viability. This data demonstrate that polymeric nanocapsules are readily internalized by T-cells and do not induce a toxic effect.
  • FSC forward light scatter
  • SSC side light scatters
  • eGFP was synthesized upon release of mRNA or pDNA from capsule shell during its degradation.
  • significantly higher transfection efficiencies were obtained with mRNA than with pDNA.
  • mRNA- and DNA-loaded capsules of both types exerted minimal cytotoxicity (viability of about 94%).
  • T- lymphocytes represent the model of hard-to-transfect cell types, such as hematopoietic stem cells. This demonstrates the proof of the concept for the synthesis of nano-sized MeCCh nanoparticles suitable for the generation of polyelectrolyte nanocapsules that efficiently load the nucleic acids and provide efficient DNA and mRNA gene delivery into clinically relevant human cells.
  • nanocapsules synthesis protocol provides a polymer capsule size about 50-100 nm, which provides a great internalization efficiency with clinically relevant cell types that are not demonstrate phagocytic activity.
  • biocompatible polyelectrolyte nanocapsules described here can be used for delivering biologically active compounds to target cells.

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Abstract

La présente invention concerne des procédés de préparation de nanoparticules et/ou de nanocapsules ayant de faibles tailles nanométriques, des compositions comprenant de telles nanoparticules et/ou nanocapsules, et leur utilisation dans l'administration de molécules (par exemple, acides nucléiques, peptides, protéines, ou petites molécules) à différents types de cellules, en particulier des types de cellules cliniquement pertinents.
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